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Improved Multiplex-PCR Method for the Simultaneous Detection of 6

Food Bacteria Producing Biogenic Amines7

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Blanca de las Rivas, Ángela Marcobal, Rosario Muñoz*10

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Departamento de Microbiología, Instituto de Fermentaciones Industriales, Juan de la 14

Cierva 3, 28006 Madrid, Spain15

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*Corresponding author. Tel.: + 34 91 562 2900; Fax: + 34 91 564 48 5320

E-mail address: rmunoz@ifi.csic.es (R. Muñoz)21

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Keywords: Multiplex-PCR; Biogenic amines; Histamine; Tyramine; Putrescine; 24

Detection method25

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Abstract1

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This study describes a simple and rapid multiplex-PCR method to determine the 3

ability to produce histamine, tyramine and putrescine by bacteria. The assay is an 4

improved method based on an assay designed by lactic acid bacteria. This improved 5

method includes a pair of primers based on sequences from histidine decarboxylases 6

from gram-negative bacteria. Under the optimised conditions, the assay yielded a 367-7

bp DNA fragment from histidine decarboxylases of gram-positive bacteria, 534-bp 8

fragment from histidine decarboxylases of gram-negative bacteria, 924-bp from 9

bacterial tyrosine decarboxylases, and 1446-bp fragment from bacterial ornithine 10

decarboxylases. The method was successfully applied to several biogenic amine-11

producing bacterial strains, even when DNAs of several target organisms were included 12

in the same reaction. This simple method could be easily incorporated in food control 13

laboratories to detect potentially biogenic amine-producing bacteria in foods.14

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1.Introduction1

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Biogenic amines are organic bases of low molecular weight, possessing 3

biological activity. Although biogenic amines such histamine, tyrosine and putrescine 4

are needed for many critical functions in man and animals, consumption of foods 5

containing high amounts of these amines can have toxicological effects. A typical 6

phenomenon is the “cheese reaction”, usually caused by high levels of tyramine in 7

cheese. However, the most notorious food-borne intoxications caused by biogenic 8

amines are related to histamine. Histamine poisoning is often referred to as “scombroid 9

fish poisoning” because of the frequent association of this illness with the consumption 10

of scombriod fish, such as tuna, mackerel and sardines [1]. Putrescine, although seems 11

to have a lower pharmacological activity than histamine and tyramine, hampers the 12

detoxification of histamine and/or tyramine. Moreover, putrescine can react with nitrite 13

to form carcinogenic nitrosamines. For this reason it is particularly important to prevent 14

the accumulation of biogenic amines in cured food products [2].15

Biogenic amines are present in a wide range of food products, including fish 16

products, meat products, dairy products, wine, beer, vegetables, fruits, and nuts [3]. In 17

non-fermented foods, the presence of biogenic amines is indicative of undesired 18

microbial activity [4]. In most amine-containing foods the majority of the amines are 19

generated by decarboxylation of the corresponding amino acids through substrate-20

specific enzymes derived from microorganisms present in the food. Although amino 21

acid decarboxylases are not widely distributed among bacteria, species of many genera 22

are capable of decarboxylating one or more amino acids. However, the ability of 23

microorganims to decarboxylate amino acids is highly variable. It depends not only on 24

the species, but also on the strain and the environmental conditions [5, 6].25

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Early detection of biogenic amine-producing bacteria is important in the food 1

industry because there are cause of food poisoning. Therefore, the use of methods for 2

the early and rapid detection of these bacteria is important for preventing biogenic 3

amine accumulation in food products. Molecular methods for detection and 4

identification of food-borne bacteria are becoming widely accepted as an alternative to 5

traditional culture methods. PCR has become an important method for the rapid, 6

sensitive, and specific detection of targeted genes. PCR techniques targeting bacterial 7

amino acid decarboxylase genes have been previously designed. Le Jeune et al. [7] 8

developed three primer sets for the detection of histamine-producing lactic acid 9

bacteria; similarly, a primer set for the detection of histamine-producing gram-negative 10

bacteria was described [8]. Recently, Coton et al. [9] described a primer set for the 11

detection of tyramine-producing lactic acid bacteria. All these methods detect bacteria 12

producing only one biogenic amine; therefore, a multiplex-PCR method for the 13

simultaneously detection of lactic acid bacteria producing histamine, tyramine and 14

putrescine was developed in our laboratory recently [10].15

While histamine in fermented products is produced by gram-positive lactic acid 16

bacteria, histamine in raw fish products is caused mostly by gram-negative enteric 17

bacteria such as Morganella morganii, Klebsiella spp., etc. Similarly, most 18

Enterobacteriaceae, gram-negative bacteria, are capable of producing putrescine in food 19

products. Therefore, our previously described multiplex-PCR method should be 20

improved in order to detect also gram-negative biogenic amine-producing bacteria in 21

foods.22

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2. Materials and methods1

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2.1. Strains and growth conditions3

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The following pure cultures of histamine-producing bacterial strains were 5

purchased from the Spanish Type Culture Collection (CECT): Klebsiella planticola6

CECT 843, Proteus vulgaris CECT 484, Photobacterium phosphoreum CECT 4192. 7

The CECT also provided Morganella morganii CECT 173, histamine and putrescine 8

producing, and the tyramine-producing Lactobacillus brevis CECT 5354. Lactobacillus9

30a (ATCC 33222), an histamine- and putrescine-producing strain was purchased from 10

the American Type Culture Collection (ATCC). An histamine producing 11

Staphylococcus sp. strain and a putrescine-producing Serratia liquefaciens strain, were 12

kindly provided by Dr. A. V. Carrascosa; both strains were isolated from meat products 13

and belong to his bacterial collection. Escherichia coli DH5α, a laboratory K-12 strain, 14

was used as putrescine-producing gram-negative strain.15

Lactic acid bacteria strains were grown in MRS broth (Difco, France) and 16

incubated at 30 ºC. Gram-negative bacteria were growth in nutritive agar (Difco, 17

France) and incubated at the temperature recommended by the CECT.18

Production of biogenic amines was tested by growing each strain in the 19

decarboxylase medium described by Maijala [11]. The medium contained the 20

corresponding precursor amino acid (L-histidine monohydrochloride, tyrosine disodium 21

salt, and L-ornithine monohydrochloride) at a 0.5% final concentration. The precursor 22

amino acids were purchased from Sigma (St. Louis, MO, USA). A positive result is 23

indicated by a change to purple in response of the indicator to a pH shift.24

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2.2. DNA manipulations and PCR procedures1

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Chromosomal DNA extraction was carried out as described elsewhere [12]. PCR 3

products were carried out in 1.5 % agarose gels and stained with ethidium bromide as 4

described [13]. PCR amplifications were performed as described previously [10]. 5

Briefly, the PCR was performed in 25-µl amplification reaction mixture containing 12.5 6

ng of template DNA, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 2.5 mM MgCl2, 200 mM 7

of each dNTP, and 1 U of AmpliTaq GoldTM DNA polymerase. The relative 8

concentration of several primers was optimised previously [10] being 0.3 µM of primer 9

set JV16HC-JV17HC, 1 µM of primer set 3-16, and 2 µM of primer set P1-rev-P2-for. 10

Primers for the amplification of the histidine decarboxylase (hdc) gene of gram-negative 11

bacteria were used in a final concentration of 1 µM. The two primers sets tested for the 12

amplification of the hdc gene in gram-negative bacteria were: primer hdc-f and hdc-r, 13

previously described [8], and primers 106 (5´-14

AAYTCNTTYGAYTTYGARAARGARG) coding for NSFDFEKEV and 107 (5´-15

ATNGGNGANCCDATCATYTTRTGNCC) coding for GHKMIGSPI, that amplified 16

DNA fragments of 709 and 534 bp, respectively.17

The reactions were performed in a GeneAmp PCR System 2400 (Perkin-Elmer) 18

using the following cycling parameters: 10 min for enzyme activation at 95 ºC followed 19

by 30 cycles of 30 sec at 95 ºC, 30 sec at 52 ºC, and 2 min at 72 ºC; and a final 20

extension step of 10 min at 72 ºC [10]. 21

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3. Results and discussion1

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3.1. Amplification of the hdc gene in gram-negative bacteria3

4We have previously described a multiplex-PCR method for the simultaneous 5

detection of lactic acid bacteria producing histamine, tyramine and putrescine [10]. It is 6

well know that gram-negative bacteria are able to produce these compounds in foods. In 7

order to extend the scope of our method by detecting also gram-negative bacteria, and 8

taking as starter method our previous multiplex-PCR assay, we planned to develop a 9

new multiplex-PCR method.10

First of all, we need to select new oligonucleotide primers for the amplification 11

of amino acid decarboxylase genes in gram-negative bacteria. Since the most frequent 12

foodborne intoxications caused by biogenic amines involves histamine, first, we 13

checked oligonucleotide primers to amplify the hdc gene in gram-negative bacteria. 14

Since histamine is the decarboxylation product of histidine, catalysed specifically by the 15

enzyme histidine decarboxylase (HDC), it is possible to develop a molecular detection 16

method that detects the gene responsible for production of this enzyme. There are two 17

distinct classes of HDCs enzymes, pyridoxal 5´-phosphate (PLP)-dependent in gram-18

negative bacteria [14] and pyruvoil-dependent in gram-positive bacteria [15, 16]. 19

Uniplex-PCR methods for the detection of genes encoding one type of HDC coding 20

genes have been developed. For gram-positive bacteria, Le Jeune et al. [7] described 21

primer set JV16HC and JV17HC that were successfully used to detect histamine-22

producing Clostridium perfringens strains [17]. Our multiplex-PCR method described 23

for lactic acid bacteria includes this primer set. For gram-negative bacteria, Takahashi et 24

al. [8] described primers hdc-f and hdc-r that amplify a 709-bp fragment of the hdc gene 25

in a high number of histamine-producing strains. Therefore, in order to select primers 26

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for the amplification of the hdc gene in gram-negative bacteria, our first approach was 1

to use Takahashi´s primers in a uniplex-PCR experiment and by using the PCR program 2

optimized for the multiplex assay. As shown in Fig. 1 A, histamine-producing bacteria 3

gave an amplicon of the expected size, however, in some strains, additional unspecific 4

amplicons could be observed. This is not surprising, since Takahashi et al. [8] 5

performed their uniplex-PCR assay at higher annealing temperature (58 ºC) than the 6

optimised for the multiplex assay (52 ºC). 7

In order to design a new pair of oligonucleotide primers for the detection of 8

gram-negative bacteria hdc gene to be used in the multiplex-PCR assay, we aligned 9

amino acid sequences of known hdc proteins from Raoultella planticola (accession 10

number P28578), Morganella morganii (P05034), and Pseudomonas fluorescens11

(P95477), among others. Two conserved domains were selected to design the synthetic 12

primers 106 and 107, that amplified a 534-bp hdc DNA fragment. When these primers 13

were used in a uniplex-PCR reaction by using the PCR program optimised for the 14

multiplex assay, only the expected 534-bp fragment was obtained (Fig. 1, B). It is 15

noteworthy that psycrophilic Photobacterium spp. could be easily detected by this 16

primer set since it has been shown to be a more dominant histamine producing than 17

Morganella morganii in fish samples stored at temperatures lower than 15 ºC [18]. 18

As deduced from these results, primers 106 and 107 might constitute a valid 19

uniplex-PCR method for the successful detection of histamine-producing gram-negative 20

bacteria. These primers seems to be more specific than Takahashi´s primers, since at the 21

same annealing temperature they gave only the specific amplicon. 22

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3.2. Amplification of the odc gene in gram-negative bacteria1

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Similarly, oligonucleotide primers need to be designed for the detection of gram-3

negative bacteria odc gene to be used in the multiplex-PCR assay. Primers 3 and 16 4

were designed previously based on amino acid sequences of well-known or putative 5

ornithine decarboxylases from gram-positive (Lactobacillus 30a, Lactobacillus 6

jhonsonii NCC533) and gram-negative bacteria (Escherichia coli, Salmonella 7

typhimurium, Vibrio cholerae, and so on). We have previously demonstrated that this 8

primer set amplified successfully lactic acid bacteria producing putrescine. However, 9

we did not tested them in gram-negative bacteria. In order to test the ability of primers 3 10

and 16 to amplify odc genes in gram-negative bacteria, we performed uniplex-PCR 11

experiments in putrescine-producing strains belonging to Morganella morganii, 12

Escherichia coli, and Serratia liquefaciens species. As expected, and by using the 13

optimised PCR program for the multiplex assay, primer set 3-16 gave only the expected 14

amplicon of 1.4 kb (data not shown).15

As far as we known, primers 3 and 16 are the only primers described in the 16

literature to be applied in a PCR method for the molecular detection of putrescine-17

producing bacteria. These primers were successfully used in the detection of the 18

putrescine-producing lactic acid bacteria, and, by using them, we identified the ornithine 19

decarboxylase gene in Oenococcus oeni BIFI-83, a putrescine-producing lactic-acid 20

bacteria [19].21

Moreover, in this study we have demonstrated that putrescine-producing gram-22

negative bacteria could be also successfully detected by primer set 3-16. 23

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3.3. PCR-multiplex detection of biogenic amine-producing bacteria1

2As mentioned above, we have previously described a multiplex-PCR method for 3

the detection of lactic acid bacteria producing histamine, tyramine and putrescine [10]. 4

In order to extend the scope of this multiplex-PCR method to gram-negative biogenic 5

amine producing strains, we need to incorporate primers sets to amplify their histidine, 6

tyrosine and ornithine decarboxylase genes. 7

As far as we known, only gram-positive bacteria have been described to produce 8

tyramine [9, 20, 21]. However, gene sequences of gram-negative bacteria sharing 9

similarity with plant tyrosine/L-DOPA decarboxylases have been discovered in the 10

complete genomes of Pseudomonas putida KT2440 (accession number NP_744697) 11

and Yersinia pestis (NP_404801). These proteins showed also high similarity to a 12

decarboxylase from the gram-negative bacteria Sorangium cellulosum So ce90 that only 13

decarboxylates L-dopa [22]; therefore, it is possible that similar proteins in gram-14

negative bacteria might possess the same specific function. 15

In order to detect gram-negative putrescine-producing bacteria, we have 16

demonstrated above that primer set 3-16 are also useful for their detection and, being an 17

advantage since they are already included in the multiplex-PCR method. Similarly, 18

primer set 106-107 have been shown to be useful for the detection of the histamine 19

producing gram-negative bacteria; however, they need to be included in the multiplex-20

PCR assay. 21

We performed PCR-experiments by including the four oligonucleotide pairs in 22

the same reaction, and by using several DNAs from biogenic amine producing strains as 23

DNA template. In a first attempt, primer set 106-107 was included in the multiplex 24

reaction at 1 µM final concentration. At this concentration, their amplicon does not 25

interfere with other amplicons, and originates a specific 534 bp hdc DNA fragment only 26

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in the putrescine-producing strains. Table 1 summarizes the results obtained by the 1

application of the improved multiplex-PCR method to biogenic amine-producing non-2

lactic acid bacteria. Figure 2 showed some of the DNA fragment obtained. All the 3

biogenic amine producing bacteria amplified DNA products of the expected sizes only 4

in presence of respective DNA template.5

In order to test the resolution of the multiplex-PCR method to amplify several 6

amplicons simultaneously, we performed PCR experiments by adding a DNA mixture 7

of Serratia liquefaciens strain (gram-negative and putrescine-producing strain), 8

Lacobacillus brevis CECT 5354 (gram-positive and tyramine-producing strain), Proteus 9

vulgaris CECT 484 (gram-negative and histamine-producing strain) and Staphylococcus10

sp. (gram-positive and histamine-producing strain) (Fig. 2). The four amplicons could 11

be easily identified in the agarose gel. Therefore, we establish this conditions as the 12

optimal for the improved multiplex assay since all the amplicons presented strong 13

intensity, the fragments sizes showed a good discrimination on agarose gel, and non-14

specific amplicons could be observed.15

16

17

In summary, we have developed an improved multiplex-PCR method for the 18

routine detection of harmful bacteria which are potential producers of histamine, 19

tyramine and putrescine in food substrates. The use of this method for the early and 20

rapid detection of biogenic-amine producing bacteria is important for preventing the 21

accumulation of histamine in food products. 22

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Acknowledgements1

2

This work was supported by grant 07G/0035/2003 from the Comunidad de 3

Madrid and RM03-002 from INIA. The technical assistance of M.V. Santamaría is 4

greatly appreciated. B. de las Rivas was a recipient of a postdoctoral fellowship and A. 5

Marcobal of a predoctoral fellowship both from the Comunidad de Madrid. 6

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References9

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[1] Lehane, L., and Olley, J. (2000). Histamine fish poisoning revisited. Int. J. Food 11

Microbiol. 58, 1-37. 12

[2] Halász, A., Barath, A., Simon-Sarkadi, L., and Holzapfel, W. (1994). Biogenic 13

amines and their production by microorganisms in foods. Trends Food Sci. 14

Technol. 5, 42-49.15

[3] Silla, M.H. (1996). Biogenic amines: their importance in foods. Int. J. Food 16

Microbiol. 29, 213-231.17

[4] Ten Brink, B., Damink, C., Joosten, H.M.L.J., and Huis in´t Veld, J.H.J. (1990). 18

Ocurrence and formation of biologically active amines in foods. Int. J. Food 19

Microbiol. 11, 73-84.20

[5] Coton, E., Rollan, G., Bertrand, A. and Lonvaud-Funel, A. (1998a). Histamine-21

producing lactic acid bacteria in wines: early detection, frequency, and distribution. 22

Am. J. Enol. Vitic. 49, 199-204.23

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[6] Veciana-Nogués, M.T., Bover-Cid, S., Mariné-Font, A., and Vidal-Carou, M.C. 1

(2004). Biogenic amine production by Morganella morganii and Klebsiella 2

oxytoca in tuna. Eur. Food Res. Technol. 218, 284-288.3

[7] Le Jeune, C., Lonvaud-Funel, A., Ten Brink, B., Holstra, H., and van der Vossen, 4

J.M.B.M. (1995). Development of a detection system for histidine decarboxylating 5

lactic acid bacteria based on DNA probes, PCR and activity test. J. Appl. Bacteriol. 6

78, 316-326.7

[8] Takahashi, H., Kimura, B., Yoshikawa, M. and, Fujii, T. (2003). Cloning and 8

sequencing of the histidine decarboxylase genes of gram-negative, histamine-9

producing bacteria and their application in detection and identification of these 10

organisms in fish. Appl. Environ. Microbiol. 69, 2568-2579.11

[9] Coton, M., Coton, E., Lucas, P., and Lonvaud, A. (2004). Identification of the gene 12

encoding a putative tyrosine decarboxylase of Carnobacterium divergens 508. 13

Development of molecular tools for the detection of tyramine-producing bacteria. 14

Food Microbiol. 21, 125-130.15

[10] Marcobal, A., de las Rivas, B., Moreno-Arribas, M.V., and Muñoz, R. Multiplex-16

PCR method for the simultaneous detection of histamine, tyramine and putrescine 17

producing lactic acid bacteria in foods. J. Food Prot. (in press) 18

[11] Maijala, R.L. (1993). Formation of histamine and tyramine by some lactic acid 19

bacteria in MRS-broth and modified decarboxylation agar. Lett. Appl. Microbiol. 20

17, 40-43.21

[12] Vaquero, I., Marcobal, A., and Muñoz, R. (2004). Tannase activity by lactic acid 22

bacteria isolated from grape must and wine. Int. J. Food Microbiol. 96, 199-204.23

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[13] Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular cloning: a 1

Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Pres, Cold Spring 2

Harbor, NY.3

[14] Kamath, A.V., Vaaler, G.L., and Snell E.E. (1991). Pyridoxal phosphate-dependent 4

histidine decarboxylases. Cloning, sequencing, and expression of genes from 5

Klebsiella planticola and Enterobacter aerogenes and properties of the 6

overexpressed enzyme. J. Biol. Chem. 266, 9432-9437.7

[15] Coton, E., Rollan, G.C., and Lonvaud-Funel, A. (1998b). Histidine carboxylase of 8

Leuconostoc oenos 9204: purification, kinetic properties, cloning and nucleotide 9

sequence of the hdc gene. J. Appl. Microbiol. 84, 143-151.10

[16] Konagaya, Y., Kimura, B., Ishida, M., and Fujii, T. (2002). Purification and 11

properties of a histidine decarboxylase from Tetragenococcus muriaticus, a 12

halophilic lactic acid bacterium. J. Appl. Microbiol. 92, 1136-1142.13

[17] Alves, R.T., dos Santos, A.T., and Martins, M.F. (2002). Detection of histamine-14

producing bacteria using polymerase chain reaction techniques and DNA probes. 15

Eur. Food Res. Technol. 214, 178-180.16

[18] Kanki, M., Yoda, T., Ishibashi, M., and Tsukamoto, T. (2004). Photobacterium 17

phoshoreum caused a histamine fish poisoning incident. Int. J. Food Microbiol. 92, 18

79-87. 19

[19] Marcobal, A., de las Rivas, B., Moreno-Arribas, M.V., and Muñoz, R. (2004). 20

Identification of the ornithine decarboxylase gene in the putrescine-producer 21

Oenococcus oeni BIFI-83. FEMS Microbiol. Lett. 239, 213-220.22

[20] Connil, N., Le Breton, Y., Dousset, X., Auffray, Y., Rince, A., and Prevost, H. 23

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involved in tyramine production. Appl. Environ. Microbiol. 68, 3537-3544.25

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[21] Lucas, P., Landete, J., Coton, M., Coton, E., and Lonavud-Funel, A. (2003). The 1

tyrosine decarboxylase operon of Lactobacillus brevis IOEB 9809. characterization 2

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71.4

[22] Müller, R., Gerth, K., Brandt, P., Blöcker, H. and Beyer, S. (2000). Identification 5

of an L-dopa decarboxylase gene from Sorangium cellulosum So ce90. Arch. 6

Microbiol. 173, 303-306. 7

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Figure legends1

2

Fig. 1. Uniplex-PCR amplification of the histidine decarboxylase gene (hdc) in gram-3

negative bacteria by using primers hdc-f and hdc-r that give an amplicon of 709 bp (A) 4

or primers 106 and 107 giving an amplicon of 534 bp (B). PCR was performed by 5

using the conditions described in the Material and methods section by using DNA from 6

Photobacter phosphoreum CECT 4192 (lane 1), Proteus vulgaris CECT 484 (lane 2), 7

Klebsiella planticola CECT 843 (lane 3) or Morganella morganii CECT 173 as DNA 8

template. The molecular sizes (in kilobases) of some standards (EcoRI and HindIII 9

digested λ DNA) are indicated on the left.10

11

Fig. 2. Multiplex-PCR detection of bacteria producing histamine, tyramine and 12

putrescine. The multiplex-PCR assay was performed by including simultaneously the 13

following primers pairs JV16HC and JV17HC at 0.3 µM final concentration (amplicon 14

size 367 bp), 106 and 107 at 1 µM (amplicon size 534 bp), P1-rev and P2-for at 2 µM 15

(amplicon size 924 bp) and 3 and 16 at 1 µM (amplicon size 1446 bp) in the reaction. 16

The reaction was performed by using chromosomal DNA from Serratia liquefaciens17

(lane 1), Staphylococcus sp. (lane 2), Lactobacillus brevis CECT 5354 (lane 3), 18

Lactobacillus 30a (lane 4), Proteus vulgaris CECT 484, and a DNA mixture of four 19

DNAs extracted from Serratia liquefaciens, Lactobacillus brevis CECT 5354, Proteus 20

vulgaris CECT 484 and Staphylococcus sp. (lane 6). The molecular sizes (in kilobases) 21

of some standards (EcoRI and HindIII digested λ DNA) are indicated on the left. A 50 22

bp leader marker was also included on the right. 23

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Table 1. Application of the multiplex-PCR method to biogenic amine-producing non-lactic acid bacteria.

Target gene and amplicon size (bp)a

hdc tdc odcBacterial species 367 534 924 1446Klebsiella planticola - + - -Proteus vulgaris - + - -Photobacterium phosphoreum - + - -Morganella morganii - + - +Escherichia coli - - - +Serratia liquefaciens - - - +Staphylococcus sp. + - - -

ahdc, histidine decarboxylase gene amplified with primer set JV16HC-JV17HC (367 bp) or primer set 106-107 (534 bp); tdc, tyrosine decarboxylase gene amplified with primer set P1-rev-P2-for; and, odc, ornithine decarboxylase gene amplified with primers 3 and 16.

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Figure 1

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Figure 2

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